Cd47 targeted therapies for the treatment of infectious disease

ABSTRACT

Methods are provided for treating a subject with for an intracellular pathogen infection, by administering an agent that reduces the binding of CD47 on a infected cell to SIRPα on a host phagocytic cell, in an effective dose for increasing the phagocytosis of infected cells.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 16/902,068, filed Jun. 15, 2020, which is a Continuation ofapplication Ser. No. 16/214,507, filed Dec. 10, 2018, now U.S. Pat. No.10,723,803, issued Jul. 28, 2020, which is a Continuation of applicationSer. No. 15/676,296, filed Aug. 14, 2017, now U.S. Pat. No. 10,184,004,issued Jan. 22, 2019, which is a Continuation of application Ser. No.14/763,758, filed Jul. 27, 2015, now U.S. Pat. No. 9,771,428, issuedSep. 26, 2017, which is a 371 application and claims the benefit of PCTApplication No. PCT/US2014/014905, filed Feb. 5, 2014, which claimsbenefit of U.S. Provisional Patent Application No. 61/761,133, filedFeb. 5, 2013, which applications are incorporated herein by reference intheir entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contractsCA168059, GM007365, and HHSN266200700002C awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Turnover of cells begins with the induction of an apoptotic program orother cellular changes that mark them for removal, and the subsequentrecognition of markers by phagocytes, including macrophages, dendriticcells, and the like. This process requires a specific and selectiveremoval of unwanted cells. Unlike healthy cells, the unwanted/aged/dyingcells display markers or ligands called “eat-me” signals, i.e. “alteredself”, which can in turn be recognized by receptors on the phagocytes.Healthy cells may display “don't eat-me” signals that actively inhibitphagocytosis; these signals are either downregulated in the dying cells,are present in an altered conformation or they are superseded by theupregulation of “eat-me” or pro-phagocytic signals. The cell surfaceprotein CD47 on healthy cells and its engagement of a phagocytereceptor, SIRPα, constitutes a key “don't eat-me” signal that can turnoff engulfment mediated by multiple modalities, including apoptotic cellclearance and FcR mediated phagocytosis. Blocking the CD47 mediatedengagement of SIRPα on a phagocyte, or the loss of CD47 expression inknockout mice, can cause removal of live cells and non-agederythrocytes. Alternatively, blocking SIRPα also allows engulfment oftargets that are not normally phagocytosed, for those cells wherepre-phagocytic signals are also present.

CD47 is a broadly expressed transmembrane glycoprotein with a singleIg-like domain and five membrane spanning regions, which functions as acellular ligand for SIRPα with binding mediated through the NH2-terminalV-like domain of SIRPα. SIRPα is expressed primarily on myeloid cells,including macrophages, granulocytes, myeloid dendritic cells (DCs), mastcells, and their precursors, including hematopoietic stem cells.Structural determinants on SIRPα that mediate CD47 binding are discussedby Lee et al. (2007) J. Immunol. 179:7741-7750; Hatherley et al. (2007)J.B.C. 282:14567-75; and the role of SIRPα cis dimerization in CD47binding is discussed by Lee et al. (2010) J.B.C. 285:37953-63.

In keeping with the role of CD47 to inhibit phagocytosis of normalcells, there is evidence that it is transiently upregulated onhematopoietic stem cells (HSCs) and progenitors just prior to and duringtheir migratory phase, and that the level of CD47 on these cellsdetermines the probability that they are engulfed in vivo. CD47 is alsoconstitutively upregulated on a number of cancers. Overexpression ofCD47 by tumor cells may increase pathogenicity by allowing the cell toevade phagocytosis.

Programmed cell death (PCD) and phagocytic cell removal are common waysthat damaged, precancerous, inflamed, or infected cells respond topathogenic threats to the organism. However, some infections persist forlong periods of time, suggesting that successful persistent infectionsovercome the PCD and phagocytic cell removal pathways. Theidentification and targeting of a mechanism by which infectious agentsovercome PCD and/or phagocytic cell removal will prove useful for thetreatment of infectious disease via disruption of the identifiedmechanism. The present invention provides methods for treatment ofinfectious disease using CD47 blocking reagents.

SUMMARY OF THE INVENTION

Methods are provided for treating an individual infected with anintracellular pathogen, by administering an effective dose of an agentthat reduces the binding between CD47 present on a cell infected withthe intracellular pathogen, to SIRPα on a phagocytic cell present in theindividual, where the dose is effective in increasing the phagocytosisof infected cells. Suitable agents include soluble high affinity SIRPαpolypeptides; soluble CD47; anti-CD47 antibodies, anti-SIRPα antibodies,and the like. Antibodies of interest include humanized antibodies, orcaninized, felinized, equinized, bovinized, porcinized, etc.,antibodies, and variants thereof.

Embodiments of the invention include treating a mammalian subject,including without limitation dog, cat, pig, sheep, cow, horse, human,etc. Embodiments of the invention include treating a subject for anyintracellular pathogen infection. In particular embodiments the methodsare used in the treatment of chronic pathogen infections, for exampleincluding but not limited to viral infections, e.g. retrovirus,lentivirus, hepadna virus, herpes viruses, pox viruses, human papillomaviruses, etc.; intracellular bacterial infections, e.g. Mycobacterium,Chlamydophila, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella,Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, etc.; andintracellular protozoan pathogens, e.g. Plasmodium sp, Trypanosoma sp.,Giardia sp., Toxoplasma sp., Leishmania sp., etc.

In some embodiments, the methods provided are for targeting or depletinginfected cells, comprising contacting a population of cells, e.g. bloodfrom an infected subject, with an agent that specifically binds to CD47,in order to target or deplete infected cells. In certain aspects, theagent is an anti-CD47 antibody or high affinity soluble SIRPα conjugatedto a cytotoxic agent, e.g., radioactive isotope, chemotherapeutic agent,toxin, etc. In some embodiments, the depletion is performed on an exvivo population of cells (e.g., the purging of infected cells from thesubject's blood). In another embodiment, methods are provided for invivo targeting of infected cells in a subject by administering such anagent to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A-1D demonstrates an upregulation of CD47 in Friend Virus(FV)-infected cells compared to uninfected cells from the same animal (7days post-infection of adult mice). (FIG. 1A and FIG. 1B) CD19+ B cells(FIG. 1C and FIG. 1D) Ter119+ Erythroid cells. “34+” is a marker ofvirus-infected cells while “34−” indicates uninfected cells.

FIG. 2A-2B demonstrates an upregulation of CD47 in Fr98-infected vsuninfected cells from the same animal (mouse neonates). “34+” is amarker of virus-infected cells while “34−” indicates uninfected cells.Fr98 is a neurotropic mouse leukemia virus.

FIG. 3A-3B demonstrates an upregulation of CD47 in individual cell typesthat are infected with the Fr98 neurotropic mouse leukemia virus. (FIG.3A) indicates the percent of each cell type that stained positive formAb34+ when infected with virus compared to a mock infection. (FIG. 3B)indicates the level of CD47 expression per cell (MFI—Mean FluorescenceIntensity) for infected versus uninfected cells from the same animal.Multiple different cell types were assayed.

FIG. 4A-4D demonstrates an increase of CD47 expression upon HIVinfection of human cells. (FIG. 4A-4B) T cells were isolated from peopleand either (FIG. 4A) HIV infected or (FIG. 4B) mock infected. Thepercent of cells expressing p24 antigen was determined by FACS. (FIG.4C-4D) demonstrate increased CD47 expression on HIV-infected T cellsrelative to uninfected T cells within a single sample. The two graphs inFIG. 4C represent duplicate experiments. The two graphs in FIG. 4Drepresent duplicate experiments.

FIG. 5 demonstrates increased CD47 levels in La Crosse Virus infectedcells compared to uninfected cells for a variety of different cell types(28 days post-infection of mice).

FIG. 6A-6B demonstrates an upregulation of CD47 in HeLa cells infectedwith Chlamydia serovar A, a strain with reduced macrophage tropism. CD47expression was detected by staining with recombinant SIRPα-Fc fusionprotein.

FIG. 7A-7D presents an evaluation of the antiviral activity of anti-CD47humanized monoclonal antibody 5F9-hIgG4 against established HIV-1(JR-CSF) infection in SCID-hu Thy/Liv mice treated by intraperitonealinjection. (FIG. 7A) summary of experimental protocol. (FIG. 7B)indicates HIV p24 antigen levels and HIV-1 RNA copy number followingcontrol or anti-CD47 treatment as indicated. (FIG. 7C) depicts flowcytometric analysis of human thymocytes following control or anti-CD47treatment as indicated. (FIG. 7D) shows absolute cell counts and bodyweight change from treated animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of treating a subject for aninfection by administering an agent that reduces the binding of CD47 toSIRPα, which may be referred to herein as an anti-CD47 agent.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect can be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom. Those in need of treatment include those already with aninfection as well as those in which an infection is to be prevented. Assuch, a therapeutic treatment is one in which the subject is infectedprior to administration and a prophylactic treatment is one in which thesubject is not infected prior to administration. In some embodiments,the subject is suspected of being infected prior to administration. Insome embodiments, the subject has an increased risk of infection priorto administration. In some embodiments, the subject is suspected ofbeing at increased risk of infection prior to administration.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and refer to any mammalian subject forwhom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc.Preferably, the mammal is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. For purposes of this invention, an effectiveamount of an anti-CD47 agent is an amount that is sufficient topalliate, ameliorate, stabilize, reverse, prevent, slow or delay theprogression of the disease state (e.g., infection) by increasingphagocytosis of a target cell.

As used herein, a “target cell” is a cell expressing CD47 on thesurface, where masking or otherwise altering the CD47 positive phenotype(e.g., by administration of an anti-CD47 agent) results in increasedphagocytosis. Usually a target cell is a mammalian cell, for example ahuman cell.

As used herein, the term “infection” refers to any state in at least onecell of an organism (i.e., a subject) is infected by an infectiousagent. As used herein, the term “infectious agent” refers to a foreignbiological entity, i.e. a pathogen, that induces increased CD47expression in at least one cell of the infected organism. For example,infectious agents include, but are not limited to bacteria, viruses,protozoans, and fungi. Intracellular pathogens are of particularinterest. Infectious diseases are disorders caused by infectious agents.Some infectious agents cause no recognizable symptoms or disease undercertain conditions, but have the potential to cause symptoms or diseaseunder changed conditions.

As used herein, the term “anti-CD47 agent” refers to any agent thatreduces the binding of CD47 (e.g., on an infected cell) to SIRPα (e.g.,on a phagocytic cell). Non-limiting examples of suitable anti-CD47reagents include high affinity SIRPα reagents, anti-SIRPα antibodies,soluble CD47 polypeptides, and anti-CD47 antibodies or antibodyfragments. In some embodiments, a suitable anti-CD47 agent (e.g. ananti-CD47 antibody, a high affinity SIRPα reagent, etc.) specificallybinds CD47 to reduce the binding of CD47 to SIRPα. In some embodiments,a suitable anti-CD47 agent (e.g., an anti-SIRPα antibody, a soluble CD47polypeptide, etc.) specifically binds SIRPα to reduce the binding ofCD47 to SIRPα. A suitable anti-CD47 agent that binds SIRPα does notactivate SIRPα (e.g., in the SIRPα-expressing phagocytic cell). Theefficacy of a suitable anti-CD47 agent can be assessed by assaying theagent (further described below). In an exemplary assay, infected cellsare incubated in the presence or absence of the candidate agent. Anagent for use in the methods of the invention will up-regulatephagocytosis by at least 10% (e.g., at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, at least 120%, at least 140%, at least 160%, atleast 160%, or at least 200%) compared to phagocytosis in the absence ofthe agent. Similarly, an in vitro assay for levels of tyrosinephosphorylation of SIRPα will show a decrease in phosphorylation by atleast 5% (e.g., at least 10%, at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100%) compared to phosphorylation observed in absence ofthe candidate agent.

In some embodiments, the anti-CD47 agent does not activate CD47 uponbinding. When CD47 is activated, a process akin to apoptosis (i.e.,programmed cell death) occurs (Manna and Frazier, Cancer Research, 64,1026-1036, Feb. 1, 2004). Thus, in some embodiments, the anti-CD47 agentdoes not directly induce cell death of a CD47-expressing cell.

The terms “specific binding,” “specifically binds,” and the like, referto non-covalent or covalent preferential binding to a molecule relativeto other molecules or moieties in a solution or reaction mixture (e.g.,an antibody specifically binds to a particular polypeptide or epitoperelative to other available polypeptides, or high affinity binding of aSIRPα polypeptide). In some embodiments, the affinity of one moleculefor another molecule to which it specifically binds is characterized bya K_(D) (dissociation constant) of 10⁻⁵ M or less (e.g., 10⁻⁶ M or less,10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, 10⁻¹⁵ M orless, or 10⁻¹⁶ M or less). “Affinity” refers to the strength of binding,increased binding affinity being correlated with a lower K_(D).

The term “specific binding member” as used herein refers to a member ofa specific binding pair (i.e., two molecules, usually two differentmolecules, where one of the molecules, e.g., a first specific bindingmember, through non-covalent means specifically binds to the othermolecule, e.g., a second specific binding member). Suitable specificbinding members include agents that specifically bind CD47 (i.e.,anti-CD47 agents), or that otherwise block the interaction between CD47and SIRPα.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

In one embodiment of the invention, the anti-CD47 agent, or apharmaceutical composition comprising the agent, is provided in anamount effective to detectably inhibit the binding of CD47 to SIRPαpresent on the surface of phagocytic cells. The effective amount isdetermined via empirical testing routine in the art, for example in abiological sample taken from an infected individual. The effectiveamount may vary depending on the number of cells being targeted, thelocation of the cells, and factors specific to the subject.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “sample” with respect to a patient encompasses blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas cancer cells. The definition also includes sample that have beenenriched for particular types of molecules, e.g., nucleic acids,polypeptides, etc.

The term “biological sample” encompasses a clinical sample, and alsoincludes tissue obtained by surgical resection, tissue obtained bybiopsy, cells in culture, cell supernatants, cell lysates, tissuesamples, organs, bone marrow, blood, plasma, serum, and the like. A“biological sample” includes a sample obtained from a patient's infectedcell, e.g., a sample comprising polynucleotides and/or polypeptides thatis obtained from a patient's infected cell (e.g., a cell lysate or othercell extract comprising polynucleotides and/or polypeptides); and asample comprising infected cells from a patient. A biological samplecomprising an infected cell from a patient can also include non-infectedcells.

High affinity SIRPα reagent. In some embodiments, a subject anti-CD47agent is a “high affinity SIRPα reagent”, which includes SIRPα-derivedpolypeptides and analogs thereof. High affinity SIRPα reagents aredescribed in international application PCT/US13/21937, which is herebyspecifically incorporated by reference. High affinity SIRPα reagents arevariants of the native SIRPα protein. In some embodiments, a highaffinity SIRPα reagent is soluble, where the polypeptide lacks the SIRPαtransmembrane domain and comprises at least one amino acid changerelative to the wild-type SIRPα sequence, and wherein the amino acidchange increases the affinity of the SIRPα polypeptide binding to CD47,for example by decreasing the off-rate by at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 500-fold, ormore.

A high affinity SIRPα reagent comprises the portion of SIRPα that issufficient to bind CD47 at a recognizable affinity, e.g., high affinity,which normally lies between the signal sequence and the transmembranedomain, or a fragment thereof that retains the binding activity. Thehigh affinity SIRPα reagent will usually comprise at least the d1 domainof SIRPα with modified amino acid residues to increase affinity. In someembodiments, a SIRPα variant of the present invention is a fusionprotein, e.g., fused in frame with a second polypeptide. In someembodiments, the second polypeptide is capable of increasing the size ofthe fusion protein, e.g., so that the fusion protein will not be clearedfrom the circulation rapidly. In some embodiments, the secondpolypeptide is part or whole of an immunoglobulin Fc region. In otherembodiments, the second polypeptide is any suitable polypeptide that issubstantially similar to Fc, e.g., providing increased size,multimerization domains, and/or additional binding or interaction withIg molecules.

A suitable high affinity SIRPα reagent reduces (e.g., blocks, prevents,etc.) the interaction between the native proteins SIRPα and CD47. Theamino acid changes that provide for increased affinity are localized inthe d1 domain, and thus high affinity SIRPα reagents comprise a d1domain of human SIRPα, with at least one amino acid change relative tothe wild-type sequence within the d1 domain. Such a high affinity SIRPαreagent optionally comprises additional amino acid sequences, forexample antibody Fc sequences; portions of the wild-type human SIRPαprotein other than the d1 domain, including without limitation residues150 to 374 of the native protein or fragments thereof, usually fragmentscontiguous with the d1 domain; and the like. High affinity SIRPαreagents may be monomeric or multimeric, i.e. dimer, trimer, tetramer,etc.

Anti-CD47 antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds CD47 (i.e., an anti-CD47 antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). In someembodiments, a suitable anti-CD47 antibody does not activate CD47 uponbinding. Non-limiting examples of suitable antibodies include clonesB6H12, 5F9, 8B6, and C3 (for example as described in InternationalPatent Publication WO 2011/143624, herein specifically incorporated byreference).

Anti-SIRPα antibodies. In some embodiments, a subject anti-CD47 agent isan antibody that specifically binds SIRPα (i.e., an anti-SIRPα antibody)and reduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). Suitableanti-SIRPα antibodies can bind SIRPα without activating or stimulatingsignaling through SIRPα because activation of SIRPα would inhibitphagocytosis. Instead, suitable anti-SIRPα antibodies facilitate thepreferential phagocytosis of infected cells over non-infected cells.Those cells that express higher levels of CD47 (e.g., infected cells)relative to other cells (non-infected cells) will be preferentiallyphagocytosed. Thus, a suitable anti-SIRPα antibody specifically bindsSIRPα without activating/stimulating enough of a signaling response toinhibit phagocytosis.

Soluble CD47 polypeptides. In some embodiments, a subject anti-CD47agent is a soluble CD47 polypeptide that specifically binds SIRPα andreduces the interaction between CD47 on one cell (e.g., an infectedcell) and SIRPα on another cell (e.g., a phagocytic cell). A suitablesoluble CD47 polypeptide can bind SIRPα without activating orstimulating signaling through SIRPα because activation of SIRPα wouldinhibit phagocytosis. Instead, suitable soluble CD47 polypeptidesfacilitate the preferential phagocytosis of infected cells overnon-infected cells. Those cells that express higher levels of CD47(e.g., infected cells) relative to other cells (non-infected cells) willbe preferentially phagocytosed. Thus, a suitable soluble CD47polypeptide specifically binds SIRPα without activating/stimulatingenough of a signaling response to inhibit phagocytosis.

In some cases, a suitable soluble CD47 polypeptide can be a fusionprotein (for example as structurally described in US Patent PublicationUS20100239579, herein specifically incorporated by reference). However,only fusion proteins that do not activate/stimulate SIRPα are suitablefor the methods provided herein. Suitable soluble CD47 polypeptides alsoinclude any peptide or peptide fragment comprising variant or naturallyexisting CD47 sequences (e.g., extracellular domain sequences orextracellular domain variants) that can specifically bind SIRPα andinhibit the interaction between CD47 and SIRPα without stimulatingenough SIRPα activity to inhibit phagocytosis.

In certain embodiments, soluble CD47 polypeptide comprises theextracellular domain of CD47, including the signal peptide (SEQ IDNO:2), such that the extracellular portion of CD47 is typically 142amino acids in length, and has the amino acid sequence set forth in SEQID NO:3. The soluble CD47 polypeptides described herein also includeCD47 extracellular domain variants that comprise an amino acid sequenceat least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% (or any percentidentity not specifically enumerated between 65% to 100%), whichvariants retain the capability to bind to SIRPα without stimulatingSIRPα signaling.

In certain embodiments, the signal peptide amino acid sequence may besubstituted with a signal peptide amino acid sequence that is derivedfrom another polypeptide (e.g., for example, an immunoglobulin orCTLA4). For example, unlike full-length CD47, which is a cell surfacepolypeptide that traverses the outer cell membrane, the soluble CD47polypeptides are secreted; accordingly, a polynucleotide encoding asoluble CD47 polypeptide may include a nucleotide sequence encoding asignal peptide that is associated with a polypeptide that is normallysecreted from a cell.

In other embodiments, the soluble CD47 polypeptide comprises anextracellular domain of CD47 that lacks the signal peptide. In anexemplary embodiment, the CD47 extracellular domain lacking the signalpeptide has the amino acid sequence set forth in SEQ ID NO:1 (124 aminoacids). As described herein, signal peptides are not exposed on the cellsurface of a secreted or transmembrane protein because either the signalpeptide is cleaved during translocation of the protein or the signalpeptide remains anchored in the outer cell membrane (such a peptide isalso called a signal anchor). The signal peptide sequence of CD47 isbelieved to be cleaved from the precursor CD47 polypeptide in vivo.

In other embodiments, a soluble CD47 polypeptide comprises a CD47extracellular domain variant. Such a soluble CD47 polypeptide retainsthe capability to bind to SIRPα without stimulating SIRPα signaling. TheCD47 extracellular domain variant may have an amino acid sequence thatis at least 65%-75%, 75%-80%, 80-85%, 85%-90%, or 95%-99% identical(which includes any percent identity between any one of the describedranges) to SEQ ID NO:1.

In some cases, an anti-CD47 agent is not a soluble CD47 polypeptide(i.e., is an anti-CD47 agent other than a soluble CD47 polypeptide). Insome cases, an anti-CD47 agent binds to SIRPα but is not a soluble CD47polypeptide (i.e., is a SIRPα binding anti-CD47 agent other than asoluble CD47 polypeptide).

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibodies” (Abs) and“immunoglobulins” (Igs) are glycoproteins having the same structuralcharacteristics. While antibodies exhibit binding specificity to aspecific antigen, immunoglobulins include both antibodies and otherantibody-like molecules which lack antigen specificity. Polypeptides ofthe latter kind are, for example, produced at low levels by the lymphsystem and at increased levels by myelomas.

“Antibody fragment”, and all grammatical variants thereof, as usedherein are defined as a portion of an intact antibody comprising theantigen binding site or variable region of the intact antibody, whereinthe portion is free of the constant heavy chain domains (i.e. CH2, CH3,and CH4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include Fab, Fab′, Fab′-SH,F(ab′)2, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv (scFv) molecules (2)single chain polypeptides containing only one light chain variabledomain, or a fragment thereof that contains the three CDRs of the lightchain variable domain, without an associated heavy chain moiety (3)single chain polypeptides containing only one heavy chain variableregion, or a fragment thereof containing the three CDRs of the heavychain variable region, without an associated light chain moiety and (4)nanobodies comprising single Ig domains from non-human species or otherspecific single-domain binding modules; and multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Suitable anti-CD47 antibodies include fully human, humanized or chimericversions of such antibodies. Humanized antibodies are especially usefulfor in vivo applications in humans due to their low antigenicity.Similarly caninized, felinized, etc antibodies are especially useful forapplications in dogs, cats, and other species respectively.

Methods

Methods are provided for treating or reducing infection, includingwithout limitation bacterial, viral, protozoan, and fungal infections,by inhibiting the interaction between SIRPα and CD47, thereby increasingin vivo phagocytosis of infected cells. Such methods includeadministering to a subject in need of treatment a therapeuticallyeffective amount or an effective dose of an anti-CD47 agent, includingwithout limitation combinations of the reagent with another drug.

In some embodiments the infection is a chronic infection, i.e. aninfection that is not cleared by the host immune system within a periodof up to 1 week, 2 weeks, etc. In some cases, chronic infections involveintegration of pathogen genetic elements into the host genome, e.g.retroviruses, lentiviruses, Hepatitis B virus, etc. In other cases,chronic infections, for example certain intracellular bacteria orprotozoan pathogens, result from a pathogen cell residing within a hostcell. Additionally, in some embodiments, the infection is in a latentstage, as with herpes viruses or human papilloma viruses.

Viral pathogens of interest include without limitation, retroviral andlentiviral pathogens, e.g. HIV-1; HIV-2, HTLV, FIV, SIV, etc., HepatitisB virus, etc. Microbes of interest, but not limited to the following,include: Yersinia sp., e.g. Y. pestis, Y. pseudotuberculosis, Yenterocolitica; Francisella sp.; Pasteurella sp.; Vibrio sp., e.g. V.cholerae, V. parahemolyticus; Legionella sp., e.g. L. pneumophila;Listeria sp., e.g. L. monocytogenes; Mycoplasma sp., e.g. M. hominis, M.pneumoniae; Mycobacterium sp., e.g. M. tuberculosis, M. leprae;Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C.trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp., e.g. H.pylori, etc. Also included are intracellular protozoan pathogens, e.g.Plasmodium sp, Trypanosoma sp., Giardia sp., Toxoplasma sp., Leishmaniasp., etc. In some cases, the pathogen is not a virus. In some cases, thepathogen is not a pox virus. In some cases, the pathogen is a virus, butis not a pox virus (i.e., the pathogen is a virus other than a poxvirus). In some cases, the pathogen is a virus, but is not a vacciniavirus (i.e., the pathogen is a virus other than a vaccinia virus). Insome cases, the pathogen is a virus, but is not a molluscum contagiosumvirus (MCV) (i.e., the pathogen is a virus other than MCV). In somecases, the pathogen is a virus, but is not a pox virus, a vacciniavirus, or a molluscum contagiosum virus (MCV) (i.e., the pathogen is avirus other than a pox virus, vaccinia virus, or MCV).

An infection treated with the methods of the invention generallyinvolves a pathogen with at least a portion of its life-cycle within ahost cell, i.e. an intracellular phase. The methods of the inventionprovide for a more effective removal of infected cells by the phagocyticcells of the host organism, relative to phagocytosis in the absence oftreatment, and thus are directed to the intracellular phase of thepathogen life cycle.

In some embodiments, the methods of the invention involve diagnosis of apatient as suffering from a pathogenic intracellular infection; orselection of a patient previously diagnosed as suffering from apathogenic intracellular infection; treating the patient with a regimenof anti-CD47 therapy, optionally in combination with an additionaltherapy; and monitoring the patient for efficacy of treatment.Monitoring may measure clinical indicia of infection, e.g. fever, whiteblood cell count, etc., and/or direct monitoring for presence of thepathogen.

Treatment may be combined with other active agents. Classes ofantibiotics include penicillins, e.g. penicillin G, penicillin V,methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.;penicillins in combination with β-lactamase inhibitors, cephalosporins,e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems;monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins;polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole;spectinomycin; trimethoprim; vancomycin; etc. Cytokines may also beincluded, e.g. interferon γ, tumor necrosis factor α, interleukin 12,etc. Antiviral agents, e.g. acyclovir, gancyclovir, etc., may also beused in treatment.

Effective doses of the therapeutic entity of the present invention varydepending upon many different factors, including the nature of theanti-CD47 agent, means of administration, target site, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but nonhuman mammals mayalso be treated, e.g. companion animals such as dogs, cats, horses,etc., laboratory mammals such as rabbits, mice, rats, etc., and thelike. Treatment dosages can be titrated to optimize safety and efficacy.

In some embodiments, the therapeutic dosage can range from about 0.0001to 500 mg/kg, and more usually 0.01 to 100 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-50 mg/kg. The dosage may be adjusted forthe molecular weight of the reagent. An exemplary treatment regimeentails administration daily, semi-weekly, weekly, once every two weeks,once a month, etc. In another example, treatment can be given as acontinuous infusion. Therapeutic entities of the present invention areusually administered on multiple occasions. Intervals between singledosages can be weekly, monthly or yearly. Intervals can also beirregular as indicated by measuring blood levels of the therapeuticentity in the patient. Alternatively, therapeutic entities of thepresent invention can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the polypeptidein the patient. It will be understood by one of skill in the art thatsuch guidelines will be adjusted for the molecular weight of the activeagent, e.g. in the use of antibody fragments, in the use of antibodyconjugates, in the use of high affinity SIRPα reagents, etc. The dosagemay also be varied for localized administration, e.g. intranasal,inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v.,and the like.

For the treatment of disease, the appropriate dosage of the anti-CD47agent will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the agent isadministered for preventive purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The anti-CD47 agent is suitably administered to thepatient at one time or over a series of treatments.

Suitable anti-CD47 agents can be provided in pharmaceutical compositionssuitable for therapeutic use, e.g. for human treatment. In someembodiments, pharmaceutical compositions of the present inventioninclude one or more therapeutic entities of the present invention orpharmaceutically acceptable salts, esters or solvates thereof. In someother embodiments, the use of an anti-CD47 agent includes use incombination with another therapeutic agent, e.g., another anti-infectionagent. Therapeutic formulations comprising one or more anti-CD47 agentsof the invention are prepared for storage by mixing the anti-CD47 agenthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. The anti-CD47 agentcomposition will be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the anti-CD47 agent to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent the CD47associated disease.

The anti-CD47 agent can be administered by any suitable means, includingtopical, oral, parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, intrathecalor subcutaneous administration. In addition, the anti-CD47 agent issuitably administered by pulse infusion, particularly with decliningdoses of the agent.

The anti-CD47 agent need not be, but is optionally formulated with oneor more agents that potentiate activity, or that otherwise increase thetherapeutic effect. These are generally used in the same dosages andwith administration routes as used hereinbefore or about from 1 to 99%of the heretofore employed dosages.

An anti-CD47 agent is often administered as a pharmaceutical compositioncomprising an active therapeutic agent and another pharmaceuticallyacceptable excipient. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

In still some other embodiments, pharmaceutical compositions can alsoinclude large, slowly metabolized macromolecules such as proteins,polysaccharides such as chitosan, polylactic acids, polyglycolic acidsand copolymers (such as latex functionalized Sepharose™, agarose,cellulose, and the like), polymeric amino acids, amino acid copolymers,and lipid aggregates (such as oil droplets or liposomes).

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group, and non-covalentassociations. Suitable covalent-bond carriers include proteins such asalbumins, peptides, and polysaccharides such as aminodextran, each ofwhich have multiple sites for the attachment of moieties. A carrier mayalso bear an anti-CD47 agent by non-covalent associations, such asnon-covalent bonding or by encapsulation. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindinganti-CD47 agents, or will be able to ascertain such, using routineexperimentation.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Carriers and linkers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. A radionuclidechelate may be formed from chelating compounds that include thosecontaining nitrogen and sulfur atoms as the donor atoms for binding themetal, or metal oxide, radionuclide.

Radiographic moieties for use as imaging moieties in the presentinvention include compounds and chelates with relatively large atoms,such as gold, iridium, technetium, barium, thallium, iodine, and theirisotopes. It is preferred that less toxic radiographic imaging moieties,such as iodine or iodine isotopes, be utilized in the methods of theinvention. Such moieties may be conjugated to the anti-CD47 agentthrough an acceptable chemical linker or chelation carrier. Positronemitting moieties for use in the present invention include ¹⁸F, whichcan be easily conjugated by a fluorination reaction with the anti-CD47agent.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above. Langer, Science 249:1527,1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. Theagents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient. The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Toxicity of the anti-CD47 agents can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. The dataobtained from these cell culture assays and animal studies can be usedin formulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without departing from the spirit or scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Examples

The data presented here demonstrate that increased expression of CD47 isa common mechanism by which infectious agents (e.g., viruses, fungi,bacteria, protozoa, etc.) circumvent the immune response of the hostorganism. When an infected cell is induced by an infectious agent toexpress increased levels of CD47, the infected cell presents a “don'teat me” signal (CD47) to the host organism's macrophages and phagocytes.Thus, the infectious agent prevents the phagocytosis and removal of theinfected cell. To counteract this process and allow the phagocytosis andremoval of infected cells in a subject, an anti-CD47 agent can beadministered to reduce the binding of CD47 on the infected cell, toSIRPα on a phagocytic host cell and thus allowing the eat me signals toprevail.

The following experiments were performed, revealing that cells infectedwith various infectious agents express higher levels of CD47 thanuninfected cells.

Adult mice were experimentally infected with the Friend Virus (FV virus,a strain of murine leukemia virus that is a member of retroviridaehaving a ssRNA genome). Uninfected and infected cells (i.e., FV-infectedcells) from the same animal were isolated 7 days post-infection andassayed via Fluorescent activated cell sorting (FACS) to determinerelative expression levels of CD47 (using an anti-CD47 antibody).FV-infected cells (B cells as well as Erythroid cells) expressed higherlevels of CD47 compared to uninfected cells from the same animal (FIG.1A-1D).

Infected and uninfected cells were isolated from mouse neonates infectedwith the Fr98-neurotropic mouse leukemia virus. FR98-infected cellsexpressed increased levels of CD47 compared to uninfected cells from thesame animal (FIG. 2A-2B). This trend held true for multiple individualcell types that were isolated, demonstrating a general trend thatinfected cells express increased levels of CD47 compared to uninfectedcells, regardless of cell type (FIG. 3A-3B).

Increased levels of CD47 were expressed by HIV-infected cells comparedto uninfected cells (FIG. 4A-4D). PBMCs were isolated from 3 differentpeople, and the cells underwent CD8-CD56-bead negative selection andstimulation with anti-CD3, anti-CD28 and IL-2 for 4 days.

Increased levels of CD47 were expressed by a variety of differentinfected cell types isolated from mice 28 days following infection withthe La Crosse Virus compared to uninfected cells (FIG. 5).

Increased levels of CD47 were expressed by HeLa cells infected withChlamydia trachomatis serovar A (a bacterial pathogen) compared touninfected cells (FIG. 6A-6B).

Based on the above findings, the binding of CD47 on a first cell toSIRPα on a second cell is expected to increase the phagocytosis ofinfected cells. To test this expectation, an anti-CD47 agent (theanti-CD47 antibody 5F9-hIgG4) was administered to mice (SCID-hu Thy/Liv)harboring an HIV infection.

As described in Stoddart et al. (Stoddart C A et al., PLoS One. 2007Aug. 1; 2(7):e655), the SCID-hu mouse model, in which human lymphoidorgans are implanted into severe-combined immunodeficient (SCID) mice,was designed to provide a small animal model for the study of humanhematopoiesis (McCune, 1988, Science 241:1632-1639). These micefacilitated study of the pathogenesis of HIV-1 in human hematolymphoidorgans and evaluation of anti-HIV-1 compounds in vivo. In this model,SCID mice are implanted with a variety of human fetal organs, includingbone, liver, thymus, lymph node, and spleen. The fetal implants becometolerant of the mouse environment, and reciprocally, growth of the humantissue is permitted by the immunocompromised status of the recipientSCID mouse.

The SCID-hu Thy/Liv mouse, first reported by Namikawa et al. in 1990 (JExp Med 172: 1055-1063,1990), is generated by co-implanting human fetalthymus and liver beneath the mouse kidney capsule. In a highlyreproducible manner, these organs fuse, become vascularized, and growinto a stable organ termed “Thy/Liv,” reaching a total mass of100-300×10⁶ human cells in 18 weeks. The Thy/Liv implant reproduces thedifferentiation, proliferation, and function of human hematopoieticprogenitor cells derived from the fetal liver within the human thymus.The implants possess histologically normal cortical and medullarycompartments that sustain multilineage human hematopoiesis for 6-12months, generating a continuous source of CD4-expressing thymocytes thatcan serve as target cells for HIV-1 infection and replication.Importantly for a model of antiviral chemotherapy, 50-60 SCID-hu Thy/Livmice can be made with tissues from a single fetal donor, and the Thy/Livimplant is amenable to experimental manipulation and infection withHIV-1. The Thy/Liv implants support viral replication after inoculationof HIV-1 by direct injection, and thymocyte depletion occurs with bothmolecular clones and clinical isolates of HIV-1 in 3-5 weeks. Thisdepletion includes loss of CD4⁺CD8⁺ (double-positive, DP) immaturecortical thymocytes and a decrease in the CD4/CD8 ratio in the thymicmedulla.

The SCID-hu Thy/Liv mouse model of HIV-1 infection is considered in theart to be a useful platform for the preclinical evaluation of antiviralefficacy in vivo. This model of HIV is considered to be a highlyreproducible mouse model that is likely to predict clinical antiviralefficacy in humans (Stoddart C A et al., PLoS One. 2007 Aug. 1;2(7):e655).

An anti-CD47 agent (a humanized monoclonal antibody 5F9-hIgG4) wasadministered via intraperitoneal injection to SCID-hu Thy/Liv miceharboring a persistent HIV infection. An evaluation of the antiviralactivity of the anti-CD47 agent is presented in FIG. 7A-7D,demonstrating the effectiveness of the methods of the invention.

1. A method of treating a mammalian subject for intracellular pathogeninfection, wherein the pathogen is a bacterium, the method comprising:administering to the subject an anti-CD47 agent that reduces the bindingof CD47 on an infected cell to SIRPα on a phagocytic cell, at aneffective dose for increasing the phagocytosis of the infected cell,wherein the anti-CD47 agent is: (a) an anti-CD47 antibody, (b) ananti-SIRPα antibody that does not stimulate signaling through SIRPα, (c)a SIRPα-derived polypeptide that specifically binds to CD47, or (d) asoluble CD47 polypeptide that specifically binds to SIRPα and does notstimulate signaling through SIRPα.
 2. The method of claim 1, wherein thesubject is a human.
 3. The method of claim 1, wherein the bacterium isselected from Chlamydia sp., Yersinia sp., Francisella sp.; Pasteurellasp.; Vibrio sp., Legionella sp., Listeria sp., Mycoplasma sp.,Mycobacterium sp., Rickettsia sp., and Helicobacter sp.
 4. The method ofclaim 1, wherein the bacterium is selected from Chlamydia sp., Chlamydiatrachomatis, Chlamydia pneumoniae, and Chlamydia psittaci.
 5. The methodof claim 1, wherein the bacterium is Chlamydia trachomatis.
 6. Themethod of claim 1 wherein the anti-CD47 agent is an anti-CD47 antibody.7. The method of claim 6, wherein the anti-CD47 antibody is a fullyhuman, humanized or chimeric antibody.
 8. The method of claim 7, whereinthe antibody is humanized 5F9-hIgG4.
 9. The method of claim 6, whereinthe anti-CD47 antibody is an antibody fragment: (i) Fab, Fab′, Fab′-SH,F(ab′)2, Fv fragment or (ii) diabody.
 10. The method of claim 1, whereinthe anti-CD47 agent is an anti-SIRPα antibody that does not stimulatesignaling through SIRPα.
 11. The method of claim 10, wherein theanti-SIRPα antibody is a humanized antibody.
 12. The method of claim 10,wherein the anti-SIRPα antibody is an antibody fragment: (i) Fab, Fab′,Fab′-SH, F(ab′)2, Fv fragment or (ii) diabody.
 13. The method of claim1, wherein the anti-CD47 agent is a SIRPα-derived polypeptide thatspecifically binds to CD47 comprising at least the d1 domain of SIRPαwith modified amino acid residues to increase affinity.
 14. The methodof claim 13, wherein the SIRPα-derived polypeptide is a SIRPα-derivedpolypeptide is fused in frame with an immunoglobulin Fc region.
 15. Themethod of claim 1, wherein the anti-CD47 agent is a soluble CD47polypeptide that specifically binds to SIRPα and does not stimulatesignaling through SIRPα.